I have extracted total RNA from Jatropha latex and I got 2856 ng/uL, A260/280 was 0.9. The buffer I used containing with 4% PVP and treated the sample with Proteinase K (heated at 40oC for 15 min) following phenol:chloroform:isoamyl alcohol extraction. The RNA was precipitated by glacial acetic acid and 2 volume of absolute ethanol. The RNA pellet was very white. How can I remove the contaminant on RNA pellet?
Hi there,
Macherey Nagel Nucleospin RNA plant kit should be helpful. For info:
http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/RNA%20and%20mRNA/UM_TotalRNAPlant.pdf
There are many great kits for RNA extraction, one of which Dominique has mentioned above.
However, should you wish to continue with your current method, here are some tips. Check to make sure your Phenol has an acid pH (less than 7) before starting. Since you are using Phenol:Chloroform:Isoamyl Alcohol, the proteins will denature and are present as a white layer at the interface between the organic and aqueous phases (your nucleic acids are in the aqueous phase) after centrifugation. You can then carefully pipette off the aqueous phase into a new tube, leaving the protein behind. Repeat this twice to ensure complete removal of proteins. There should be no need to use proteinase K unless your samples are highly proteinaceous.
Your A260/280 is very low, you should aim for around 2. If you are performing phase separation correctly, then this is likely phenol contamination - in which case wash the aqueous phase with chloroform before precipitation. http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf
You should also be treating your samples with DNAse I (RNAse-FREE) after extraction to remove the genomic DNA. Then repurifying to remove the DNAse.
Protocol as follows:
Pellet cells and aspirate media, or grind plant or animal tissue in liquid Nitrogen. Add 1 mL of resuspension buffer (20 mM NaOAc, 1 mM EDTA), 1 mL Phenol:Chloroform (plus 0.2 mg of acid washed beads if using cells). Vortex vigorously for 3 x 2 minutes. Centrifuge at 4000 rcf for 5 min. Separate aqueous phase to clean tube being careful not to disturb the protein at the interface.
Add 1 mL phenol:Chloroform to the aqueous phase (now in a new tube), vortex briefly (15 - 30 sec), then spin at full speed for 5 min. Separate aqueous phase to new tube, add equal volume of phenol, vortex (15-30 sec), spin at full speed (5 min). Separate aqueous phase to clean tube, add equal volume of chloroform (to remove phenol contamination), vortex, spin, separate aqueous phase to clean tube. Add 3 M NaOAc pH 7 (10% of the volume of the aqueous phase) and 2.5 volumes of Ethanol, precipitate at -80 for 1 hr or overnight. Spin at full speed for 30 min at 4 degrees C to pellet nucleic acid. Resuspend in suitable buffer (TE, MOPS or RNAse-free water) with RNAse inhibitor, add DNAse I (RNAse-free) plus buffer (according to Manufacturer's instruction, and incubatte 30 min at room temp, then repeat the Phenol:Chloroform extraction to remove the DNAse (since you probably dont want this in downstream applications).
Hope this helps & good luck!
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